The specific aims of the proposal are the isolation and characterization of inactive renin and renin in human amniotic fluid. Amiotic fluid will be pooled and concentrated. Ion exchange chromatography on DEAE-cellulose with non-linear gradient elution will be the initial step although phenylathylamine-agarose is being evaluated as a preparatory column. Inactive renin and renin will be isolated and separated following pepstatin-aminobutyl-agarose, octyl-agarose and Cibacron blue F3GA chromatographies. Renin will be detected by incubation with sheep renin substrate and RIA determination of formed angiotensin I; inactive renin will be determined similarly following activation by incubation with pepsin. Molecular weights of inactive renin (before and after activation) and renin will be determined on Sephacryl S-200. In preliminary studies, we have found the activation of inactive renin resulting from dialysis at pH 3.3 and 4 degrees is reversible by incubation at pH 7.4 and 37 degrees. We will evaluate reversibility of the activation following equilibrium gel filtration at pH 3.3 on Sephacryl S-200 to determine if inactive renin is renin combined with an inhibitor or is a conformational variant. Inactive renin and renin will be further characterized by isoelectric focusing, PAGE and active-site labeling. To date, all isolates have had inherent activity to generate angiotensin I from sheep and human substrate. However, if a totally inactive renin zymogen is isolated, it will be studied by incubations with enzymes of known mechanisms of proteolysis. Purified inactive renin and renin will be compared to the International Renin Standard by determinations of substrate kinetics with tetradecapeptide, sheep and human (adult male) substrate. The overall goal is an evaluation of the role of placental inactive renin and renin and maternal renin substrate in the pathogenesis of pregnancy-induced-hypertension. Using purified amniotic fluid inactive renin and renin, substrate kinetics will be determined with plasma obtained prospectively at various gestational stages from parents who did not develop hypertension and compared to similar determinations with plasma from patients who ultimately developed pregnancy-induced-hypertension.